The aim of this experiment is to determine the amount of sugar or carbohydrates in a soft drink provided by spectrophotometric (cololorimetric) method.

INTRODUCTION:

One useful and often used way of obtaining concentration of chemical in a solution, if it has color is by colorimetric method. If the analyte is not colored an appropriate reagent must be added that reacts with the analyte to produce a colored compound.

In this experiment, the method for determining concentration of sugar is based upon the color that forms when sugar reduce 3,5-dinitrosalicyclic acid(DNSA) to 3-amino-5-nitrosalicyclic acid.

This method tests for the presence of free carbonyl group (C=O), the so-called reducing sugars. This involves the oxidation of the aldehyde functional group present in, for example, glucose and the ketone functional group in fructose. Simultaneously, 3,5-dinitrosalicylic acid (DNS) is reduced to 3-amino,5-nitrosalicylic acid under alkaline conditions:

Oxidation

aldehyde group ----------> carboxyl group

Reduction

3,5-dinitrosalicylic acid ----------> 3-amino,5-nitrosalicylic acid

In this experiment sucrose is provided which is the non-reducing sugar, does not undergo reaction with 3,5-dinitrosalicyclic acid. Therefore the sucrose and complex carbohydrate must be broken down into simple sugars like glucose first. The hydrolysis can be done by boiling the sample with hydrochloric acid, and then the pH is adjusted to give a basic solution under which conditions are good for reducing sugar.

This method is a straightforward modification of the original DNS method for glucose analysis.

The sugars in the soft drink are at too high concentration for this method. So dilutions must be carried out before carrying out analysis.

The measurement of transmittance (T) is made by determining the ratio of the intensity of incident (I0) and transmitted (I) light passing through pure solvent and sample solutions as a function of wavelength. [Note: The percent transmittance (%T) is obtained by multiplication of T by 100.]

The logarithm of the reciprocal of the transmittance is called the absorbance (A),

A = log (1 / T)

According to Beer's law, the absorbance of a solution should be zero (100%T) if there is none of the absorbing species present. A blank solution that does not contain analyte being analyzed but have the same composition as the solution can be used to calibrate the machine into zero reading Absorbance (100%T). Then the machine can be used to find concentration of other solution.

The Beer-Lambert Law is only obeyed (the standard curve is linear) for reasonably dilute solutions.

Nature of graphs obeying Beer’s-Lambert law:

METHODOLOGY:

Material used:

·Cuvettes

·Test tubes

·Test tube rack

·Test tube clamp

·Two 400 ml beaker

·Mohr pipettes

·Bulb (5 and 10 ml)

·25ml volumetric pipette

·Five 100 ml volumetric flask

·Pasteur pipette and bulb

·Tissue paper

Chemical used:

·6M HCl

·2.5 M NaOH

·0.05 M 3,5-Dinitrosacyclic acid

·1000 mg/L standard sucrose solution

·Soft drink to test (non-diet ,not dark colored)

METHODS/PROCEDURES:

Preparation of sucrose standard solutions

·1000mg/L of standard solution was prepared by suitable dilution of the stock solution.

·2:10 dilution was made as follows, 2.0 ml of the stock solution was pipette into a clean 10 ml test tube and distilled water was added to calibrate mark of 10 ml. The tube was covered and shakes well to mix. In a similar fashion, 4:10, 6:10, and 8:10 dilutions were made.

·Five standards were prepared (the original stock solution and the four dilutions) as follows:

o2.0 ml of each sucrose standard solution were pipette into test tubes.

o2.0 ml of 6 M HCl was added into each test tube and placed in a boiling water bath for 10 minutes.

Therefore the concentration of sucrose in the original beverage is 75.666g/L

Discussion:

The DNS method can be applied twice to measure the individual concentrations of a mixture of glucose and sucrose. First, a small part of the original sample is consumed in measuring the glucose concentration by following the original DNS procedure. Another part of the sample is hydrolyzed and subsequently subjected to the same DNS procedure. The difference in the absorbance between the acid treated sample and the untreated sample is due to the presence of sucrose. The sucrose concentration can then be calculated from a calibration curve based on that difference in the absorbance.

The concentrations of standard solution were used to plot a graph.

The above concentrations were obtained by extrapolation of the standard graph of absorbance against concentration, since the absorbances of the sample were known but the concentrations are the ones which were not known.

The concentration of sucrose in the original beverage calculated deviate from the true value. This is due to various errors taking place in experiment. The mainly errors were due to reading absorbance on the spectrometer or during measurement of volumes. Other sources of error include variation of temperature and humidity.

CONCLUSION:

The nature of the graph is a straight line, hence obeys the Beer’s law, and the concentration of the original beverage was obtained to be 75.666g/L